1a. Objectives (from AD-416):
The objectives of the research project plan are as follows: 1) to evaluate the genotypic and biologic basis of susceptibility to experimentally induced mastitis by Gram-positive and Gram-negative bacteria; 2) to determine the relationship between mastitis and nitro-oxidative stress and develop innovative alternatives to reduce tissue damage; 3) to develop strategies for modulating somatic stem cells to promote the replacement of mammary epithelial cells damaged by mastitis.
1b. Approach (from AD-416):
To evaluate the genotypic and biologic basis of susceptibility to experimentally induced mastitis by Gram-positive and Gram-negative bacteria (Objective 1) we will: 1) evaluate the response of dairy cows with divergent estimated breeding value for somatic cell score, and presumptive differences in mastitis susceptibility and/or responsiveness, to experimental challenge with Gram-negative bacteria; 2) evaluate the mastitis susceptibility of cows that differ with regard to single nucleotide polymorphisms in proinflammatory cytokine promoters, which influence the innate immune response. To determine the relationship between mastitis and nitro-oxidative stress and to further develop innovative alternatives to reduce tissue damage (Objective 2) we will focus on the relationship between oxygen-nitrogen adducts that are generated to combat invading pathogens and damage to mammary tissue, as reflected by increased protein nitration, and develop targeted biochemical interventions to reduce generation of nitrated proteins. We will: 1) evaluate the extent of protein nitration in mammary glands of control and experimentally infected mastitic cows; 2) evaluate the capacity of ' - and d -tocopherols to limit protein nitration during experimentally-induced mastitis by virtue of their ability to neutralize oxygen-nitrogen adducts. To develop strategies for modulating somatic stem cells to promote the replacement of mammary epithelial cells damaged by mastitis (Objective 3) we will: 1) evaluate the ability of xanthosine to promote the expansion of mammary stem cells and study the genomic response to xanthosine treatment in order to uncover critical regulatory pathways and stem cell modulators; 2) evaluate potential markers for bovine mammary stem cells that we identified in previous transcriptome analyses (This will facilitate future mammary stem cell research); 3) Evaluate the ability of regulators of the p53 pathway, or other stem cell stimuli, to promote repair of mammary tissue that is damaged by mastitis.
3. Progress Report:
The ability to regulate bovine mammary stem cells provides a means to promote repair of damaged mammary tissue due to mastitis and enhance milk production efficiency. To identify mammary stem cells for manipulating their properties, we characterized the expression patterns of genes most highly expressed in individual cells isolated by laser microdissection to use as stem cell markers. Cells that expressed these markers were present in the expected locations in mammary tissue and changes in their abundance correlated with the lactation cycle and the inhibitory effect of ovariectomy. A manuscript has been drafted. While treatment of mammary glands with inosine or xanthosine increased the population of mammary stem cells, the treatment during the nonlactating pre-parturient period (dry period) did not increase milk production in the next lactation. It is unknown whether such treatment can be used to reverse the negative impact of an abbreviated dry period on milk yield. A comparison of gene expression in the mammary glands of cows and mice during the dry period was completed, which demonstrated the presence of very different mechanisms of mammary structural change between the species after milking is terminated. The process in mice is characteristic of total tissue regression while that in the cow reflects cell turnover, proliferation, and secretory activation. Results provide information essential to managing cows during this critical period. A manuscript has been drafted. Endophyte-infected fescue grass contains ergotoxins (ETx) that inhibit prolactin secretion, which is thought to reduce mammary differentiation and milk yield. Effect of feeding cows a diet containing endophyte-infected fescue around the period of calving is being evaluated. Although initial milk production in cows consuming endophyte-infected feed was reduced, milk production of treated cows exceeded that of non-treated cows. Histological analysis of mammary tissue during the dry period and next lactation was used to evaluate impact of ETx on the number and activity of secretory cells. Mammary growth and development were not affected by ETx. These data were presented at the Joint Annual Meeting of the Animal Science Societies (2013). Additionally, RNA from these mammary tissues was used to assess the impact of ETx on gene expression. Data analysis is underway. Infections can result in autodestructive mechanisms in critical tissues, which are initiated by chemical modifications to cellular proteins during inflammation. The need to stabilize tissues against these autodestructive processes largely has been overlooked in food animal species. Using a direct E. coli infection of the bovine mammary gland to initiate autodistructive processes, we significantly reduced tissue damage by co-instilling a nanoco-polomer encapsulated formulation of gamma- and delta- tocopherols (Vitamin E) that were successful in scavenging mediators of protein modification. Results suggest that novel delivery formulations of simple non-alpha tocopherols in plant oils can be effective in limiting the impact of infection stress on tissue integrity and function.
1. Identification of potential markers for mammary stem cells. The capacity to control the activities of stem cells of the mammary gland holds great potential for both human medicine and agriculture. The great limitation to exploiting the vast potential of these cells lies in the difficulty of properly identifying them. ARS scientists in Beltsville, MD recently developed criteria for identifying potential biological markers of mammary stem cells from their gene expression signatures. These markers aid in the development of means to regulate stem cells, which could be applied to solving problems ranging from breast cancer prevention and therapy, to increased milk production efficiency of dairy animals.
2. Bacterial infection and tissue stability. Among the earliest defenses of tissues to infection are the release of highly reactive, low molecular weight oxygen and nitrogen molecules. These molecules are toxic to the infecting organism and help to kill it. However, high concentrations of these reactive oxygen and nitrogen molecules cause death and destruction of the host’s own cells. As a result, much of the cost of recovery from infection is associated with healing and regenerating this damaged tissue. Plant oil-derived gamma- and delta-tocopherols (Vitamin E) can quickly interact with these cell-released reactants and bind them before they can injure cells at the infection site. These forms of tocopherols are very abundant in natural plant oils, making them inexpensive to mass produce. Therefore, we administered preparations of these tocopherols to infected animals and found that nanoencapsulation of the tocopherols enhanced their protective effects. The potential to use these preparations to treat infectious diseases of animals is being explored and could greatly improve animal health and well being.
Choudhary, R.K., Li, R.W., Clover, C.M., Capuco, A.V. 2013. Comparison of the transcriptpmes of long-tern label retaining-cells and C cells microdissected from mammary epithelium: an initial study to character potential stem/progenitor cells. Frontiers in Oncology. 3:21.